SKL Enhances Patency And Endothelialization Of Small-caliber Artificial Blood Vessels By Promoting Endothelial Cell Adhesion

Objective: This study is dedicated to building tissue-engineered bioactive small blood vessels.First,heparin and secreted Klotho(Secreted Klotho,SKL)were cross-linked with porcine acellular small intestinal submucosa(SIS),and then artificially sutured into tubular small blood vessels,which were transplanted into a New Zealand white rabbit model to replace part of the autologous carotid blood vessels.The samples were taken two or three months after the operation,and the expression of related indicators such as changes in the carotid artery wall and endothelialization were observed by Western blot,histological staining,and immunofluorescence.In order to explore the role of SKL-modified artificial blood vessels in replacing damaged blood vessels and the status of endothelialization,it provides an experimental basis for the construction of tissue-engineered small blood vessels.Methods: 1.Culture HEK293 cells in vitro and divide them into two groups,N group and SKL group.The N group was a normal group,and the empty vector was transfected.The SKL group was transfected with a plasmid carrying the SKL gene.Western Blot technology was used to detect and analyze the SKL protein in the transfected medium and cells.The HEK293 cell culture medium transfected SKL gene was collected,and the SKL protein was extracted and purified using a protein purification column.2.In vitro: Heparin and purified SKL protein were cross-linked to acellular SIS membrane,divided into three groups: SIS membrane,SIS membrane + heparin,SIS membrane + heparin + SKL,and human umbilical vein endothelial cells(HUVECs)were planted on SIS membrane.Scanning electron microscopy,cell proliferation and cytotoxicity experiments(CCK-8),Western Blot,and immunofluorescence were used to detect the effects of SKL on endothelial cell adhesion and proliferation.3.In vivo: Heparin and SKL protein were cross-linked on the SIS membrane,sewn into a tube with a diameter of 2mm,and transplanted into the carotid artery of the New Zealand white rabbit to replace the autologous artery.The experimental animals were divided into three groups,group N,SIS + heparin group,and SIS + heparin + SKL group.The N group was the normal group without any treatment;the SIS + heparin group,the SIS + heparin + SKL group were transplanted the artificial blood vessels of cross-linked heparin,heparin and SKL protein into rabbit carotid artery,and the samples were respectively taken two and three months later.By HE staining and Masson staining,the diameter changes of arterial wall and the condition of collagen fibers in the normal group and each experimental group were observed.CD31 and eNOS were used to detect endothelialization,and α-SMA was used to detect vascular smooth muscle regeneration.Results: 1.Western Blot detected the expression of SKL in HEK293 cells and culture medium,and found that the expression of SKL group was higher than that of N group.2.In vitro: Scanning electron microscopy showed that the number of endothelial cells adhering to the membrane of the SKL-treated group was the largest and the shape was the most stretched.CCK-8 experiments show that SKL is non-toxic to cells and has the fastest proliferation rate.Western Blot experiments showed that the expression levels of p-FAK,FAK,and RHOA in the SKL group were higher than those in the N group.Immunofluorescence also showed that the expression levels of FAK,Rac1,and RHOA in the SKL group were higher than those in the heparin group.3.In vivo: Ultrasound and HE results show that the vessel diameter of the heparin group has a certain expansion compared with the control group,and the diameter of the SKL group is much smaller than that of the heparin group.Masson staining also showed that the SKL group had a higher muscle-positive area and a higher amount of muscle fibers than the heparin group.Immunofluorescence showed that the expression levels of CD31,eNOS,and α-SMA in the SKL group were higher than those in the heparin group,and that the samples taken in 3 months were higher than those taken in 2 months.Conclusion: SKL-modified SIS small blood vessels can promote vascular endothelialization,which is of great significance for the construction of tissue engineering small blood vessels.